Design of Spacers To Increase The Expression of Recombinant Fusion Proteins

ABSTRACT

The present invention relates to fusion proteins. The invention specifically relates to compositions and methods of Tf-based fusion proteins that demonstrate a high-level expression of transferrin (Tf)-based fusion proteins by inserting a helical linker between two protein domains.

The present application claims the benefit of the filing date of U.S.Provisional Application No. 61/015,580 filed Dec. 20, 2007, thedisclosure of which is incorporated herein by reference in its entirety.

STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

The present invention is made, at least in part, with the support ofgrants from National Institute of Health (Grant R01 GM063647). Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates in general to fusion proteins. Inparticular, it relates to compositions and methods for increasing fusionprotein expression using alpha-helical spacers.

BACKGROUND OF THE INVENTION

Recombinant proteins are becoming an important class of therapeuticdrugs (1, 2). Many recombinant proteins such as growth hormones andhumanized monoclonal antibodies are already in clinical uses (3). One ofthe limitations for the production of therapeutic proteins in biotechindustry is the low yield of the recombinant proteins in cell culturesystems. Various approaches have been taken to improve the expressionand production of recombinant proteins from transfected mammalian cells,such as the selection of mutants (4), the use of virus-transfected cells(5), or the improvement of the culture medium (6). However, theseconventional methods suffer from various shortcomings. For example, theuse of mutants means that only certain mutants meeting the expressionrequirement may be used. This limits the range of proteins that may beexpressed. The choice of virus-transfected cells and culture medium arealso trial-and-error processes that require laborious experimentationsto optimize the conditions. Moreover, they don't always solve problemscaused due to structural features of the desired protein.

High quantities of recombinant proteins ranging from hundreds ofmilligrams to grams must be produced in order to carry out preclinicalevaluations and clinical trials (7-9). Unfortunately, potentialtherapeutic proteins with poor expression face an obstacle to make itthrough clinical trials to final approval by the FDA. Proteintherapeutics developed from recombinant hormones, growth factors andcytokines express at relatively low levels, not only increasing themanufacturing cost but also delaying further product evaluation. Somesuccessful protein therapeutics are recombinant fusion proteinsconsisting of cytokines or growth factors fused with the Fc portion ofIgG1 or immunotoxin and are expressed as single polypeptides with dualbiological activities (10,11). These therapeutic fusion proteins,including Enbrel® (TNF-RIFs-IgG1), Ontak® (IL-2/diphtheria toxin),Orencia® (CTLA-4/Fc-IgG1) and Amevive® (LFA-3/Fc-IgG1) (12), mayexperience poor expression as the fusion partners interfere with eachother for optimal translation, especially in mammalian cells. Sincemammalian cells are the preferred choice for producing some therapeuticproteins, as posttranslational modifications in these cells may beassociated with reduced immunogenicity compared to other systems (9), asimple strategy that enhances the expression of therapeutic fusionproteins in mammalian cells would be desirable.

Typically, the problem of low expression is improved by incorporatingcarbohydrate-binding module (CBM) and maltose-binding protein (MBP) asfusion partners to the target protein (13,14). However, these fusionpartners are generally removed during or after purification byintroducing peptide linkers with cleavage sites for endopeptidases suchas thrombin and factor Xa (14). Conceivably, this approach is notfeasible for large-scale production of target proteins because itrequires numerous steps of column purification and enzymatic processing,limiting the production capacity and possibly causing non-specificcleavage.

The selection of a peptide linker with the ability to maintain domainfunction of the fusion protein is becoming important (15-18). Recently,the inventors designed a helical linker with 50 amino acids using anEAAAK (SEQ ID NO: 3) helix-forming motif based on a previous study (16),and inserted the linker between granulocyte colony stimulating factor(G-CSF) and Tf moieties, leading to increased biological activity (19).Most recently, the inventors found that the insertion of the samehelical linker in Tf-fusion proteins resulted in a high-level expressionin HEK293 cells as compared to the same fusion proteins without thehelical linker. Here the inventors report the helical linker-dependentincrease of expression in two Tf-based fusion proteins, G-CSF and humangrowth hormone (hGH), and provide evidence of a high-level of expressionfor both proteins regardless the level of original expression withoutthe linker. Conceivably, this approach can be introduced and applied toother fusion proteins with limited to no expression, greatly improvingthe production yield for downstream applications.

The above-mentioned and other features of this invention and the mannerof obtaining and using them will become more apparent, and will be bestunderstood, by reference to the following description, taken inconjunction with the accompanying drawings. The drawings depict onlytypical embodiments of the invention and do not therefore limit itsscope.

SUMMARY OF THE INVENTION

In one embodiment, the invention relates to fusion proteins comprising ahelical linker between the protein domains and has an increased level ofexpression in a vector as compared to fusion proteins without a linkerbetween the protein domains.

In accordance with another embodiment, the invention relates tocompositions comprising fusion proteins comprising a helical linkerbetween the protein domains.

In a closely related embodiment, the invention relates to helicallinkers that allow for a high level of expression in a vector andimproved bioactivity of fusion proteins, when the linkers are insertedbetween the protein domains.

In another embodiment, the invention relates to methods of making fusionproteins comprising a helical linker inserted between the proteindomains.

In yet another embodiment, the invention relates to methods usinghelical linkers to increase the transfection of fusion protein vectorsand the production of the fusion protein, in a cellular expressionsystem.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. Oligonucleotide insert of the H4 linker and its correspondingamino acid sequence.

FIG. 2. Comparison of the expression of various fusion proteins usingWestern blotting. Lane 1: 50 ng Transferrin; Lane 2: 50 ng G-CSF; Lane3: transfected with pcDNA3.1; Lane 4: transfected withpcDNA3.1-sTf-G-CSF; Lane 5: transfected with pcDNA3.1-sTf-H41-G-CSF;Lane 6: transfected with pcDNA3.1-sTf-H42-G-CSF; Lane 7: transfectedwith pcDNA3.1-sTf-H42R-G-CSF (H42 with a reverse sequence); Lane 8:transfected with pcDNA3.0-sGCSF-Tfxx (LE); Lane 9: transfected withpcDNA3.0-sGCSF-H4(2)-Tfxx.

FIG. 3. High-level expression by the insertion of helical linker in bothhGH-Tf and Tf-hGH fusion proteins as analyzed by Anti-hGH Western blot.Four fusion proteins with or without the inserted helical linker,expressed in serum free media, were analyzed by Western blot using goatanti-hGH monoclonal antibody (1:1,000). The signal was detected usingHRP-conjugated rabbit anti-goat secondary antibody (1:1,000) and ECLreagents. The image was recorded and analyzed by ChemiDoc XBR (Bio-Rad).Lane 1: Tf (negative control); lane 2: hGH (10 ng); lane 3: hGH-Tf; lane4: hGH-(H4)2-Tf; lane 5: Tf-hGH; lane 6: Tf-(H4)2-hGH.

FIG. 4. High-level expression by the insertion of helical linker in bothhGH-Tf and Tf-hGH fusion proteins as analyzed by Anti-Tf Western blot.Four fusion proteins with or without the inserted helical linker,expressed in serum free media, were analyzed by Western blot using goatanti-Tf antibody (1:5,000). The signal was detected using rabbitanti-goat secondary antibody conjugated to HRP (1:1,000) and ECLreagents. The image was recorded and analyzed by ChemiDoc XBR. Lane 1:Tf (50 ng); lane 2: hGH (negative control); lane 3: hGH-Tf; lane 4:hGH-(H4)2-Tf; lane 5: Tf-hGH; lane 6: Tf-(H4)2-hGH.

FIG. 5. Helical linker insertion led to high-level expression in bothhGH-Tf and Tf-hGH fusion protein as analyzed by SDS-PAGE. Same volume ofconditioned media (5 μl) from the transfected HEK-293 cells werefractionated using SDS-PAGE, stained with Coomassie blue, and analyzedwith ChemiDoc XBR. MM: molecular weight marker; lanes 1-3: Tf control;lane 4: hGH-Tf; lane 5: hGH-(H4)2-Tf; lane 6: Tf-hGH; lane 7:Tf-(H4)2-hGH.

FIG. 6. Helical linker insertion led to high-level expression in bothGCSF-Tf and Tf-G-CSF fusion protein as analyzed by anti-G-CSF Westernblot. Fifteen-microliter-conditioned media from the transfected HEK-293cells were analyzed using anti-GCSF Western blot. Lane 1: G-CSF control;lane 2: pcDNA3.1(+) without the insert; lane 3: Tf-G-CSF; lane 4:Tf-(H4)-G-CSF; lane 5: Tf-(H4)2-G-CSF; lane 6: Tf-r(H4)2-G-CSF (fusionprotein with a reversed (H4)2 DNA sequence inserted, thus a non-helicallinker in the product).

DETAILED DESCRIPTION

Recently, a peptide linker, A(EAAAK)_(n)A, has been reported to form analpha-helix conformation, which is able to control the distance andreduce the interference between the domains of a recombinant protein(20). It was reported that the helix linker could effectively separatebifunctional domains of the fusion protein (21,22). The inventors of thepresent invention have previously demonstrated that the intrinsicbioactivity of the fusion protein can be optimized by the insertion ofalpha-helical spacers between G-CSF and Tf domains (23). In thisinvention, the inventors have further discovered that insertion of thealpha-helical spacers can increase either transfection or production ofthe fusion protein. Accordingly, based on the discoveries of the presentinvention, the inventors have devised methods for enhancing andoptimizing transfection of fusion protein vectors and the production ofthe fusion protein, in a cellular expression system.

As used herein the term “fusion protein” refers to or describes aprotein that comprises at least two protein domains that are separatedby one or more helical linkers. The helical linker may be EAAAK based.Examples of proteins that may used in the “fusion protein” include butare not limited to carrier proteins such as transferrin, serum albumin,an antibody or sFv, and the like, and therapeutic proteins such asinterferon, colony stimulating factor (CSF), interferon, a cytokine, ahormone, a lymphokine, an interleukin, a hematopoietic growth factor, atoxin, and the like. The fusion proteins may be expressed in a varietyof host cells, including human kidney cells, E. Coli, bacteria, yeast,and various higher eucaryotic cells such as the COS, CHO, HeLa andmyeloma cell lines, and the like. More preferably, the fusion proteincomprises at least one carrier protein and a therapeutic protein.

Materials and Methods

Preparation of Gene Fusion Constructs in pcDNA3.1(+)

Fusion constructs for Tf-based fusion proteins containing either hGH orhuman G-CSF were designed and established in the pcDNA3.1(+)(Invitrogen) mammalian expression vector based on a previous report(24). Briefly, the DNA sequences encoding for hGH or G-CSF weresubcloned and fused in frame to the sequences encoding for Tf. The DNAsequences encoding for the signal peptide from the N-terminus domainwere incorporated in the polypeptide; however, the stop codon from theN-terminus domain was deleted for uninterrupted translation. The finalconstructs were verified by DNA sequence analysis.

Helical Linker Insertion

Two domains between Tf and hGH or G-CSF in the fusion protein were fusedby leucine (L) and glutamic acid (E), a product of XhoI restrictionsite. The helical linker, H4 and (H4)₂, LEA(EAAAK)₄ALE (SEQ ID NO: 1)and LEA(EAAAK)₄ALEA(EAAAK)₄ALE (SEQ ID NO: 2), respectively, wereprepared and inserted according to the previous study (19). Theorientation, sequences and copy numbers of the helical linker wereconfirmed by DNA sequence analysis.

Production of Fusion Protein

The human embryonic kidney 293 cells (HEK293 or HEK293T;ATCC) werecultured in DMEM media (Mediatech) containing 10% FBS, 50 unitspenicillin/50 μg streptomycin in a humidified incubator at 37° C. with5% CO2. HEK293 cells were seeded at near confluence in six-well plates(Costar) and transiently transfected with 2 μg expression constructs and5.5 μl Lipofectamine 2000 (Invitrogen). The transfected cells wereallowed to express fusion proteins in serum free CD293 media(Invitrogen) for 5 days. The conditioned media containing the fusionprotein was then harvested, clarified by centrifugation, andconcentrated using Amicon Ultra-4 or Ultra-15 filtering devices(Millipore).

SDS-PAGE and Western Blots

The fusion proteins were fractionated on a 10% SDSPAGE or 4-20% pre-castgel (Thermo Scientific) and visualized by staining with Coomassie blue.For Western blot analysis, the fusion proteins were transferred to aPVDF membrane (GE healthcare) and blocked with 5% non-fat milk for 1 hat room temperature, after separating on SDSPAGE. hGH and Tf wereidentified by using goat anti-hGH monoclonal antibody (1:1,000; R&DSystems) and goat antihuman Tf antibody (1:5,000) as primary antibodiesand rabbit anti-goat antibody conjugated to HRP as secondary antibody.Likewise, the G-CSF was detected by using rabbit anti-human G-CSF(1:10,000) as primary antibody and donkey anti-rabbit antibodyconjugated to HRP (1:10,000) as secondary antibody. All antibodies wereobtained from Sigma, unless mentioned otherwise. ECL plus reagents (GEHealthcare) and ChemiDoc XBR (Bio-Rad) were used for developing andcapturing the hGH-fusion proteins. X-ray film was used to developG-CSF-fusion proteins. The expression of both hGH-and G-CSF-fusionproteins were analyzed using Quantity One software (Bio-Rad), andresults from either anti-hGH or anti-G-CSF Western blots are comparableto that of anti-Tf Western blots.

In Vitro Cell Proliferation

Nb2 cells (Sigma) derived from rat T lymphoma cells were cultured assuspension in RPMI 1640 media (Mediatech) supplemented with 2 mMglutamine, 10% FBS, 10% horse serum (HS; Invitrogen), 50 units ofpenicillin/50 pg streptomycin, and 50 μM 2-mercaptoethanol (25) in ahumidified incubator at 37° C. with 5% CO2. For proliferation assays,Nb2 cells were washed extensively in a serum free RPMI 1640 media,re-suspended in assay media that included 10% HS but not FBS, andcounted with a Z1 Coulter particle counter (Beckman Coulter). About5,000 Nb2 cells per well were seeded into 96-well plates in 200 μl assaymedia, starved for 24 h, treated with hGH or fusion protein whose dosewas normalized to that of hGH, and incubated for 4 days. Next, the cellswere added with 20 μl of Alamar Blue dye (Biosource) and incubatedovernight for color development. The UV absorbance was measured at 570nm using a Genios spectrophotometer (Tecan) and corrected by subtractingthe control without treatment. The ED50 was defined as the dose offusion protein that led to half of maximum proliferation.

The following examples are intended to illustrate, but not to limit, thescope of the invention. While such examples are typical of those thatmight be used, other procedures known to those skilled in the art mayalternatively be utilized. Indeed, those of ordinary skill in the artcan readily envision and produce further embodiments, based on theteachings herein, without undue experimentation.

EXAMPLES

Comparison of the Expression Level of Tf-G-CSF and G-CSF-Tf FusionProteins with or without H4 Linker

Construction of Plasmid

Human G-CSF fused in frame with Tf was constructed into the expressionvector pcDNA3.0. Three different peptide linkers were inserted betweenG-CSF and Tf as spacers: amino acid alpha-helical linker with 2 copiesof A(EAAAK)4A (H4)₂, a short LE dipeptide linker, and a linker with thereverse of oligonucleotide sequence of H4 to serve as a random peptidelinker.

HEK293T Cell Transient Transfection

HEK293T cells grown in monolayer were transfected with the plasmids byLipofectamine 2000. The fusion protein released into the 4-dayconditioned medium was collected.

Comparison of the Expression Level of Tf-H4n-G-CSF (n=0-2) FusionProtein

Plasmids with various copies of the H4 linker were transfected intoHEK293T cells to produce the fusion proteins. Cells were seeded into6-well culture plate 1 day prior to transfection. 2 μg of each plasmidwas transfected into each well of cells. 96 hours after transfection,the medium was collected and centrifuged at 1500 rpm for 5 min. toremove cells. 20 μl of each sample is taken to perform Western Blotting.

FIG. 2 shows comparison of the expression of various fusion proteins byusing Western blotting. The lanes in the blot are as follows:

Expression Ratio Lane 1: 50 ng Transferrin Lane 2: 50 ng G-CSF Lane 3:transfected with pcDNA3.1 Lane 4: transfected with pcDNA3.1-sTf-G-CSF 1Lane 5: transfected with pcDNA3.1-sTf-H4₁-G-CSF 7.83 Lane 6: transfectedwith pcDNA3.1 -sTf-H4₂-G-CSF 11.20 Lane 7: transfected withpcDNA3.1-sTf-H4₂R-G-CSF 0 Lane 8: transfected with pcDNA3.0-sGCSF-Tfxx(LE) 10.75 Lane 9: transfected with pcDNA3.0-sGCSF-H4(2)-Tfxx 15.52

Results

The H4 linker boosted the expression level of Tf-G-CSF. It is believedthat this effect may be achieved through increasing the stability of thefusion protein. The expression level of Tf-G-CSF fusion proteincontaining 2 copies of H4 linker (FIG. 2, lane 6) is higher than that ofTf-G-CSF containing 1 copy of H4 linker (FIG. 2, lane 5) which, in turn,is higher than that of TfG-CSF without linker (FIG. 2, lane 4).Similarly, the expression level of G-CSF-Tf containing 2 copies of H4linker (FIG. 2, lane 9) is higher than that of a short dipeptide LElinker (FIG. 2, lane 8). There was no expression of the fusion proteinwhen the inserted oligonucleotide was (H4)₂ in reverse sequence, (H4)₂R(FIG. 2, lane 7). Since the reverse sequence will generate a peptidespacer with random conformation, this finding suggests that analpha-helical structure in the spacer is required to promote theexpression and production of the fusion protein.

Gene Fusion Constructs

To investigate whether the insertion of a helical linker between theprotein domains in the Tf-based fusion protein improves the expression,the inventors constructed three pairs of gene fusion plasmids with orwithout the inserted helical linker, and confirmed that the insertion,orientation and number of copies were correct. Subsequently, the plasmidconstructs were transfected to HEK293 or HEK293T cells to produce fusionproteins.

Comparison of hGH-Tf and hGH-(H4)2-Tf Fusion Proteins for Expression

The hGH-Tf fusion protein consists of human growth hormone and Tf linkedby a short di-peptide linker; whereas the hGH-(H4)₂-Tf fusion protein islinked by a helical linker with 50 amino acids. To assess the level ofexpression, the fusion proteins with or without the helical linker wereanalyzed by Western blot as well as SDS-PAGE with Coomassie stain. Bothanti-hGH and anti-Tf Western blots detected a band corresponding toapproximately 100 kDa, which is the sum of molecular weights from Tf (79kDa) and hGH (22 kDa), and confirmed that the fusion protein wascomposed of two moieties including Tf and hGH (FIG. 4, lanes 3 and 4).To evaluate and compare the level of expression, band-densities for eachfusion protein were quantified and analyzed. The density data revealedthat the fusion protein with the helical linker expressed at a level 1.7fold higher than the original fusion protein without the helical linker(Table I, FIG. 3, lanes 3 and 4). In addition, fusion proteins wereanalyzed by SDSPAGE stained with Coomassie blue to confirm results fromthe band-density analysis from Western blots, and to evaluate relativepurity and abundance. The results from SDS-PAGE demonstrated that thefusion proteins were expressed with high purity (˜90%) and highabundance (˜95%) at a molecular weight of 100 kDa (FIG. 5, lane 5).Furthermore, band-density analysis from Coomassie blue-stained SDSPAGEshowed that the expression of fusion protein with the helical linker wasabout 1.7 fold higher than the original fusion protein without thehelical linker (FIG. 5, lane 4), confirming results from Western blotanalysis.

Comparison of Tf-hGH and Tf-(H4)2-hGH Fusion Proteins for Expression

Thus far, the fusion proteins evaluated were oriented to have aN-terminal hGH domain and C-terminal Tf domain. To investigate theeffect of the helical linker on the expression of fusion proteins withdifferent orientation, the Tf-and hGH-domains were switched to producetwo new fusion proteins: Tf-hGH and Tf-(H4)2-hGH. The two fusionproteins were expressed and the yield of expression was compared. TheWestern blot data with fusion protein specific antibodies includinganti-hGH and anti-Tf confirmed the identity and molecular weight (100kDa) of the fusion protein, as well as the insertion of helical linkeras shown by slightly increased molecular weight (FIG. 3, lanes 5 and 6,FIG. 4, lanes 5 and 6). The band-density analysis from Western blot datashowed the fusion protein with the inserted helical linker expressed ata higher level with a ˜2.4 fold increase as compared to the originalfusion protein without the helical linker (Table I). The SDS-PAGE withCoomassie stain revealed that both fusion proteins were expressed withhigh purity (˜90%) and high abundance (90%) but the fusion protein withthe helical linker expressed at high levels with a ˜2.4 fold increasecompared to the original fusion protein without the helical linker (FIG.5, lane 7 and 6, respectively), further confirming the Western blotdata.

Comparison of Tf-G-CSF and Tf-(H4)2-G-CSF Fusion Proteins for Expression

To study the broader implication of the helical linker insertion on theincreased expression of Tf-based fusion proteins, Tf-G-CSF fusionprotein consisting of transferrin and granulocyte colony-stimulatingfactor was constructed, and subsequently, the helical linker wasinserted. These two fusion proteins were expressed to compare the levelof expression. The Western blot using anti-G-CSF antibody detected a100-kDa band equivalent to the molecular weight of Tf-G-CSF fusionprotein (˜80+19 kDa), confirming the identity and molecular weight (FIG.6). Moreover, the Western blot data revealed that the expression fromthe Tf-G-CSF fusion protein was too low to detect convincingly (FIG. 6,lane 3). In contrast, the Tf-(H4)-G-CSF and Tf-(H4)₂-G-CSF fusionproteins expressed at elevated levels with a 7.8 and an 11.2 foldincrease as compared to original fusion protein without the linker,respectively, providing notable evidence for the improved expression ofTf-based fusion proteins by the insertion of a helical linker (FIG. 6,lanes 4 and 5, Table I). The fusion protein expressed with a reversedcoding sequence for the helical linker (Tf-r(H4)₂-G-CSF) failed toexpress (FIG. 6, lane 6). Taken together, the helical linker insertionbetween Tf and G-CSF domains led to a significant increase inexpression, comparing with the original fusion protein without thehelical linker.

Comparison of In Vitro Cell Proliferation

The hGH-(H4)₂-Tf and Tf-(H4)₂-hGH fusion proteins were tested in vitroto determine whether they induce Nb2 cell proliferation. The Nb2 cellstreated with either hGH-(H4)₂-Tf or Tf-(H4)₂-hGH fusion proteindemonstrated higher proliferation activity as compared to Nb2 cellstreated with the hGHTf fusion protein without helical linker (Table II).

TABLE I The Ratio of Expression of Tf-fusion Proteins with or withoutthe Helical Linker G-CSF Fusion Proteins hGH Fusion Proteins Linker (x)Tf-x-G-CS G-CSF-x-Tf Tf-x-hGH hGH-x-Tf No Liker 1 1 1 1 H4 7.8 ND ND ND(H4)₂ 11 1.44 2.39 1.66 x - linker inserted between Tf and hGH or G-CSF;(H4)₂ - two copies of helical linker; no linker - two domains were fusedby LE peptide; ND—not determined.

TABLE II Comparing Nb2 Proliferation Activity of hGH-Tf Fusion Protein,with or without the Helical Linker ED50 (ng/mL) Linker (x) hGH hGH-x-TfTf-x-hGH No Liker 0.25 1.85 NA^(a) (H4)₂ — 0.85 0.80 ^(a)Was notdetermined due to low yield of the fusion protein

Discussion

The success of constructing biologically active recombinant G-CSF-Tffusion protein with the helical linker (19,24) led us to pursue thefeasibility of producing other Tf fusion proteins. To achieve this goal,the inventors constructed recombinant fusion protein consisting of Tfand hGH, and introduced two copies of a helical linker (H4)2 as a spacerbetween two protein domains. As previously reported in G-CSF fusionprotein (19), the insertion of the helical linker also increases the invitro biological function of hGH fusion protein as shown in the Nb2 cellproliferation assay (Table II).

Besides the increase of in vitro biological activity, the inventorsfound that the expression level of both fusion proteins with the helicallinker, i.e., G-CSF-(H4)₂-Tf and hGH-(H4)₂-Tf, was significantly higherthan that of fusion proteins without the linker. The inventors furtherinvestigated the effect of helical linker insertion on the expression offusion proteins with a Tf domain switch, i.e., Tf-(H4)₂-G-CSF andTf-(H4)₂-hGH, which created a fusion protein with a differentorientation. As shown in FIGS. 5 and 6, the expression of the fusionprotein with Tf at the amino-terminus is generally very poor. However,the insertion of the helical linker significantly increased theexpression of both fusion proteins with Tf at the amino-terminus (TableI).

The inventors have reported previously that the expression of GCSF-Tffusion protein failed when a flexible and non-helical linker, consistingof glycine and serine (GSSSS)3, was inserted between Tf and G-CSFdomains (19). Others have also found that the insertion of IgG hingeregion as a flexible linker between Tf and nerve growth factor (NGF)domains was ineffective for the expression of this fusion protein (18).To further demonstrate the requirement of the helical structure of thelinker peptide for the increase of the fusion protein expression, theinventors reversed the DNA sequence coding for the helical peptide toproduce a linker with an identical peptide length, but a non-helicalstructure. Our results showed that there was no expression of the fusionprotein, Tf-r(H4)₂-GCSF (FIG. 6, lane 6). These findings led us to theconclusion that the increase of the expression of Tf-based fusionprotein is due to the non-flexible and helical nature of the linker thatis inserted between the two protein domains.

The exact mechanism responsible for this increased expression isunclear. However, the rigid, extended nature, as well as thecomposition, of the helical linker may help increase the rate of hGH-andTf-domain folding. A proper folding will enhance the stability of thenewly translated polypeptide and, consequently, will increase theexpression of Tf fusion proteins in the conditioned media. By usingfluorescent resonance energy transfer (FRET) technique, Arai et al.found that the insertion of a helical linker between the enhanced greenfluorescent protein (EGFP) domain and enhanced blue fluorescent protein(EBFP) domain in a chimeric protein increased the distance and kept twodomains apart (16). Furthermore, Robinson and Sauer (26) reported thatthe composition and length of linker between two domains were importantin controlling the rate of folding, unfolding, and stability of chimericprotein. It is likely that a large molecule, such as Tf-G-CSF or Tf-hGHfusion protein with a molecular weight of 100 kDa, requires largeconformational space to fold correctly. The stable and efficient foldingmay drive the equilibrium towards an increased expression andaccumulation of the fusion protein. Conceivably, the linker with ahelical structure can hold the domains at a distance, providing a largerspace for correct folding.

Another possible reason for the increased expression is that a helicallinker with its secondary structure may be resistant to enzymaticcleavage by protecting the target amino acids from protease recognition,thereby increasing overall stability of the fusion protein (27). Furtherstudies that evaluate the stability of the helical linker against commonproteolytic enzymes including trypsin and chymotrypsin, would aidpartially in our understanding of the mechanisms responsible for theincreased expression.

In general, a high level expression of fusion proteins may be achieved:(1) by designing expression vectors with strong promoters and episomalorigins; (2) by transfecting host cells such as HEK293T and HEK293 EBNA1with expression of episomal antigens including SV40 large-T antigen andEBV nuclear antigen, respectively; (3) by selecting transfectionreagents with highest gene transfer efficiency; and (4) by developing orusing culture media with enhanced cell proliferation and survival butwith minimum cell death and apoptosis (7-9). Furthermore, the additionof sodium butyrate and peptone into the culture media containing thetransfected cells can have positive effect on the expression of targetprotein. However, long-term consequences of using such components inculture media remain controversial and unknown (28,29). Results in thisreport demonstrate that a high level expression of Tf-fusion proteinscan be achieved by the insertion of a helical peptide linker between thetwo protein domains. If the increase of expression can be demonstratedin fusion proteins other than Tf, it will provide a simple and practicaltechnique to improve the production of recombinant fusion proteins fortherapeutic and diagnostic uses.

Conclusions

The inventors found that the insertion of a helical peptide as thelinker in Tf-based fusion proteins led to a high level of expression,with superior in vitro bioactivity. This approach provides a simplemethod to increase poor expression of other fusion proteins. Given thestraightforward approach and ease of both designing and introducing thehelical linker in the fusion protein, this qualifies as a feasiblestrategy for the production of therapeutic fusion proteins at highlevels in mammalian cells.

Methods according to the present invention can be easily adapted forcurrently established cell culture systems for recombinant proteinproduction, without changing either the cell types or the cultureconditions.

Many modifications and variation of the invention as hereinbefore setforth can be made without departing from the spirit and scope thereofand therefore only such limitations should be imposed as are indicatedby the appended claims.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

REFERENCES

1. J. M. Reichertand C. Paquette. Therapeutic recombinant proteins:trends in US approvals 1982 to 2002. Curr Opin Mol Ther 5: 139-47(2003).

2. J. M. Reichertand C. Paquette. Clinical development of therapeuticrecombinant proteins. Biotechniques 35: 176-8, 180, 182-5 (2003).

3. L. M. Weiner, Fully human therapeutic monoclonal antibodies. JImmunother 29: 1-9 (2006).

4. R. J. Kaufman. Selection and coamplification of heterologous genes inmammalian cells. Methods Enzymo/185: 537-566 (1990).

5. R. B. DuBridge, P. Tang, H. C. Hsia, P. M. Leong, J. H. Miller and M.P. Calos. Analysis of mutation in human cells by using an Epstein-barrvirus shuttle system. Mol. Cell Bioi 7: 379-387 (1987).

6. P. L. Pham, S. Perret, B. Cass, E. Carpentier, G. St-Laurent, L.Bisson, A. Kamen and Y. Durocher. Transient gene expression in HEK293cells: peptone addition posttransfection improves recombinant proteinsynthesis. Biotechnol Bioeng 90: 332-344 (2005).

7. L. Baldi, D. L. Hacker, M. Adam, and F. M. Wurm. Recombinant proteinproduction by large-scale transient gene expression in mammalian cells:state of the art and future perspectives. Biotechnol. Lett. 29:677-684(2007).

8. F. Wurm, and A. Bernard. Large-scale transient expression inmammalian cells for recombinant protein production. Curr. Opin.Biotechnol. 10:156-159 (1999).

9. F. M. Wurm. Production of recombinant protein therapeutics incultivated mammalian cells. Nat. Biotechnol. 22:1393-1398 (2004).

10. G. M. Subramanian, M. Fiscella, A. Lamouse-Smith, S. Zeuzem, and J.G. McHutchison. Albinterferon alpha-2b: a genetic fusion protein for thetreatment of chronic hepatitis C. Nat. Biotechnol. 25:1411-1419 (2007).

11. C. Wu, H. Ying, C. Grinnell, S. Bryant, R. Miller, A. Clabbers, S.Bose, D. McCarthy, R. R. Zhu, and L. Santora. Simultaneous targeting ofmultiple disease mediators by a dual-variable domain immunoglobulin.Nat. Biotechnol. 25:1290-1297 (2007).

12. B. Leader, Q. J. Baca, and D. E. Golan. Opinion: proteintherapeutics: a summary and pharmacological classification. NatureReviews Drug Discovery. 7:21-39 (2008).

13. M. Kavoosi, A. L. Creagh, D. G. Kilburn, and C. A. Haynes. Strategyfor selecting and characterizing linker peptides for CBM9-tagged fusionproteins expressed in Escherichia coli. Biotechnol. Bioeng. 98:599-610(2007).

14. K. D. Pryor, and B. Leiting. High-level expression of solubleprotein in Escherichia coli using a His6-Tag and maltose-binding proteindouble-affinity fusion system. Protein Expr. Purif. 10:309-319 (1997).

15. Y. Maeda, H. Ueda, J. Kazami, G. Kawano, E. Suzuki, and T. Nagamune.Engineering of functional chimeric protein Gvargula luciferase. Anal.Biochem. 249:147-152 (1997).

16. R. Arai, H. Ueda, A. Kitayama, N. Kamiya, and T. Nagamune. Design ofthe linkers which effectively separate domains of a bifunctional fusionprotein. Protein Eng. 14:529-532 (2001).

17. R. Arai, W. Wriggers, Y. Nishikawa, T. Nagamune, and T. Fujisawa.Conformations of variably linked chimeric proteins evaluated bysynchrotron X-ray small-angle scattering. Proteins Structure Functionand Bioinformatics. 57:829-838 (2004).

18. E. Park, R. M. Starzyk, J. P. McGrath, T. Lee, J. George, A. J.Schutz, P. Lynch, and S. D. Putney. Production and characterization offusion proteins containing transferrin and nerve growth factor. J. DrugTarget. 6:53-64 (1998).

19. Y. Bai, and W. C. Shen. Improving the oral efficacy of recombinantgranulocyte colony-stimulating factor and transferrin fusion protein byspacer optimization. Pharm. Res. 23:2116-2121 (2006).

20. R. Arai, H. Ueda, A. Kitayama, N. Kamiya, and T. Nagamune. Design ofthe linkers which effectively separate domains of a bifunctional fusionprotein. Protein Eng 14: 52932 (2001).

21. N. Jullien, F. Sampieri, A. Enjalbert, and J. P. Herman. Regulationof Cre recombinase by ligand-induced complementation of inactivefragments. Nucleic Acids Res 31: e131 (2003).

22. M. Maeda, K. Kawasaki, Y. Mu, H. Kamada, Y. Tsutsumi, T. J. Smith,and T. Mayumi. Amino acids and peptides. XXXIII. A bifunctionalpoly(ethylene glycol) hybrid of lamininrelated peptides. Biochem BiophysRes Commun 248: 485-9 (1998).

23. Y. Bai and we Shen. Improving the oral efficacy of recombinantgranulocyte colonystimulating factor and transferring fusion protein byspacer optimization. Pharm Res 23: 2116-2121 (2006).

24. Y. Bai, D. K. Ann, and W.-C. Shen. Recombinant granulocytecolony-stimulating factor-transferrin fusion protein as an oralmyelopoietic agent. Proc. Natl. Acad. Sci. 102:7292-7296 (2005).

25. M. Ishikawa, A. Nimura, R. Horikawa, N. Katsumata, O. Arisaka, M.Wada, M. Honjo, and T. Tanaka. A novel specific bioassay for serum humangrowth hormone. J. Clin. Endocrinol. Metab. 85:4274-4279 (2000).

26. C. R. Robinson, and R. T. Sauer. Optimizing the stability ofsingle-chain proteins by linker length and composition mutagenesis.Proc. Natl. Acad. Sci. 95:5929-5934 (1998).

27. S. Marqusee, and R. L. Baldwin. Helix stabilization by Glu-\cdotsLys+ salt bridges in short peptides of de novo design. Proc. Natl. Acad.Sci. U.S.A. 84:8898-8902 (1987).

28. P. L. Pham, S. Perret, B. Cass, E. Carpentier, G. St-Laurent, L.Bisson, A. Kamen, and Y. Durocher. Transient gene expression in HEK293cells: peptone addition posttransfection improves recombinant proteinsynthesis. Biotechnol. Bioeng. 90:332-344 (2005).

29. C. K. Crowell, Q. Qin, G. E. Grampp, R. A. Radcliffe, G. N. Rogers,and R. I. Scheinman. Sodium butyrate alters erythropoietin glycosylationvia multiple mechanisms. Biotechnol. Bioeng. 99:201-213 (2008).

1-6. (canceled)
 7. A fusion protein comprising a helical linker betweenthe protein domains, wherein said protein has an increased level ofbioactivity as compared to fusion proteins without said linker.
 8. Thefusion protein according to claim 7, wherein said fusion proteincomprises more than one helical linker.
 9. The fusion protein accordingto claim 7, wherein said helical linker comprises LEA(EAAAK)₄ALE (SEQ IDNO: 1) or LEA(EAAAK)₄ALEA(EAAAK)₄ALE (SEQ ID NO: 2).
 10. The fusionprotein according to claim 7, wherein said fusion protein comprises acarrier protein selected from the group comprising transferrin, serumalbumin, an antibody or sFv, and the like, and a therapeutic proteinselected from the group comprising colony stimulating factor (CSF),interferon, a cytokine, a hormone, a lymphokine, an interleukin, ahematopoietic growth factor, a toxin, and the like.
 11. The fusionprotein according to claim 10, wherein said fusion protein comprises thegranulocyte stimulating factor (G-CSF) or the human growth hormone(hGH).
 12. The fusion protein according to claim 7, wherein said proteinis (Tf)-based
 13. A helical linker comprising an EAAAK helix-formingmotif that allows for a high level of expression and improvedbioactivity of fusion proteins, when inserted between the proteindomains.
 14. The helical linker according to claim 13, wherein thesequence comprises LEA(EAAAK)₄ALE (SEQ ID NO: 1) orLEA(EAAAK)₄ALEA(EAAAK)₄ALE (SEQ ID NO: 2).
 15. A method of making fusionproteins comprising helical linkers between the protein domainscomprising (a) transfecting a construct comprising the sequences forsaid fusion proteins into cells, and (b) culturing and collecting saidfusion proteins from said cells, wherein said fusion protein has anincreased level of expression and bioactivity as compared to fusionproteins without said linker.
 16. The method according to claim 13,wherein said fusion protein comprises a carrier protein selected fromthe group comprising transferrin, serum albumin, an antibody or sFv, andthe like, and a therapeutic protein selected from the group comprisingcolony stimulating factor (CSF), interferon, a cytokine, a hormone, alymphokine, an interleukin, a hematopoietic growth factor, a toxin, andthe like.
 17. The method according to claim 15, wherein said fusionprotein is (Tf)-based.
 18. The method according to claim 13, whereinsaid construct comprises more than one helical linker.
 19. The methodaccording to claim 13, wherein said helical linker comprisesLEA(EAAAK)₄ALE (SEQ 1D NO: 1) or LEA(EAAAK)₄ALEA(EAAAK)₄ALE (SEQ ID NO:2).
 20. The method according to claim 13, wherein said cells are humanembryonic kidney cells, E. Coli, bacteria, yeast, COS, CHO, HeLa cellslines, myeloma cell lines, or any higher eucaryotic cell.